PBP Target Profiling by β-Lactam and β-Lactamase Inhibitors in Intact Pseudomonas aeruginosa: Effects of the Intrinsic and Acquired Resistance Determinants on the Periplasmic Drug Availability

ABSTRACT The lack of effective treatment options against Pseudomonas aeruginosa is one of the main contributors to the silent pandemic. Many antibiotics are ineffective against resistant isolates due to poor target site penetration, efflux, or β-lactamase hydrolysis. Critical insights to design optimized antimicrobial therapies and support translational drug development are needed. In the present work, we analyzed the periplasmic drug uptake and binding to PBPs of 11 structurally different β-lactams and 4 β-lactamase inhibitors (BLIs) in P. aeruginosa PAO1. The contribution of the most prevalent β-lactam resistance mechanisms to MIC and periplasmic target attainment was also assessed. Bacterial cultures (6.5 log10 CFU/mL) were exposed to 1/2× PAO1 MIC of each antibiotic for 30 min. Unbound PBPs were labeled with Bocillin FL and analyzed using a FluorImager. Imipenem extensively inactivated all targets. Cephalosporins preferentially targeted PBP1a and PBP3. Aztreonam and amdinocillin bound exclusively to PBP3 and to PBP2 and PBP4, respectively. Penicillins bound preferentially to PBP1a, PBP1b, and PBP3. BLIs displayed poor PBP occupancy. Inactivation of oprD elicited a notable reduction of imipenem target attainment, and it was to a lesser extent in the other carbapenems. Improved PBP occupancy was observed for the main targets of the widely used antipseudomonal penicillins, cephalosporins, meropenem, aztreonam, and amdinocillin upon oprM inactivation, in line with MIC changes. AmpC constitutive hyperexpression caused a substantial PBP occupancy reduction for the penicillins, cephalosporins, and aztreonam. Data obtained in this work will support the rational design of optimized β-lactam-based combination therapies against resistant P. aeruginosa infections. IMPORTANCE The growing problem of antibiotic resistance in Gram-negative pathogens is linked to three key aspects, (i) the progressive worldwide epidemic spread of multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR) Gram-negative strains, (ii) a decrease in the number of effective new antibiotics against multiresistant isolates, and (iii) the lack of mechanistically informed combinations and dosing strategies. Our combined efforts should focus not only on the development of new antimicrobial agents but the adequate administration of these in combination with other agents currently available in the clinic. Our work determined the effectiveness of these compounds in the clinically relevant bacteria Pseudomonas aeruginosa at the molecular level, assessing the net influx rate and their ability to access their targets and achieve bacterial killing without generating resistance. The data generated in this work will be helpful for translational drug development.

1) The amount of PBP bound by a particular beta-lactam was determined by the remaining PBP molecules available to react with Bocillin FL, in comparison to those bound by Bocillin FL in the absence of the beta-lactam. While the authors presented the gels showing the PBPs bound by Bocillin, there did not seem to be a control experiment demonstrating that the use of betalactam does not change the amount of total protein for a particular PBP. 2) AmpC hyperexpression caused noticeable MIC increases for some beta-lactams but not others. Does this pattern agree with the different activities of AmpC on these beta-lactams in previous biochemical studies? 3) Although all these PBPs bind to beta-lactams, their contributions to P. aeruginosa survival vary. Do the results in this paper suggest any PBPs are more valuable antibiotic targets than others? Do they agree with previous studies on these PBPs, including both the in vitro IC50s using purified proteins (if available) and whether they are essential? 4) Line 88, does 'the same PBP-binding affinities (IC50)' apply to one PBP or against all PBPs from the same bacteria? As the authors have shown, there are many PBPs that bind to each beta-lactam usually with varied affinities. 5) Table 1, footnote a, the listing of 'PBP4' in the knockout strains is out of order compared with the columns in the table.
Reviewer #3 (Comments for the Author): The authors compare inhibition of binding of a fluorescent penicillin toP. aeruginosa PBPs by a set of representative β-lactam antibiotics and β-lactamase inhibitors. The experimental design unfortunately limits the conclusions that can be drawn and some revision is required to take these limitations into account.
(1) The decision to use exposure at a fixed (half-MIC ) concentration and time point makes interpretation of the various observed levels of PBP inhibition difficult. Since the rate of influx and the rate of reaction with PBP at low concentration are directly proportional to inhibitor concentration, performing the experiment at concentrations that differ by as much as 500 fold (e.g. meropenem and mecillinam) means that the reactions in very different conditions are being compared and therefore conclusions about rate of entry are very uncertain. Possibly, as performed, the experiments tell more about levels of occupancy that are necessary to elicit growth inhibition by the different antibiotics. The experiments would have been better performed with different exposure times and inhibitor concentrations to assess kinetics. In this respect, the experiments with the BLIs, performed at the same concentration, are more interesting. It would be good to use a separate panel in Fig. 1, with a more useful scale, for this data set.
(2) Line 165. Has the Bocillin FL assay been calibrated for P. aeruginosa PBPs? Are the chosen concentration and incubation time appropriate to saturate all the PBPs? In the absence of this information it is difficult to interpret the data presented in Fig. 3. It appears that, for a particular PBP, the degree of labelling is similar in the different strains (except ΔdacB) but comparison of levels between PBPs is uncertain. For example, what is the significance of the difference between PBP1b and PBP2 labelling? Are the different levels really due to different amounts of protein or is the labelling of PBP2 less complete because of slower reaction or lower affinity?
(3) Line 204. The assay used does not provide information about affinity: it is only possible to say that the reaction had a greater or a lesser extent in the particular reaction conditions used for each inhibitor and with each PBP. The rates of entry and reaction as well as affinity for target all play a role in the extent of inhibition but separating these requires a more comprehensive approach, as outlined above. Reference to affinity should be deleted throughout.
(4) L. 378. There is no "mechanistic data", in terms of descriptions of rates and affinities, and this statement should be modified.

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Reviewer comments:
Reviewer #1 (Comments for the Author): The manuscript investigates the binding of beta-lactams and beta-lactam inhibitors to periplasmic PBPs in P. aeruginosa PAO1 WT and knockout strains impacting various drug resistance mechanisms (porin, beta-lactamase, and efflux). The results offer insightful new information for understanding PBP interactions with different beta-lactams, including the influences of the most common drug resistance mechanisms against these antibiotics. The concise manuscript is well written. Some minor revisions are needed.
1) The amount of PBP bound by a particular beta-lactam was determined by the remaining PBP molecules available to react with Bocillin FL, in comparison to those bound by Bocillin FL in the absence of the beta-lactam. While the authors presented the gels showing the PBPs bound by Bocillin, there did not seem to be a control experiment demonstrating that the use of beta-lactam does not change the amount of total protein for a particular PBP.
In this manuscript we wanted to highlight the PBP occupancy of the different βlactams and BLIs in intact cells, and how the different resistance mechanisms interfere with the overall target binding balance. However in another manuscript that is currently in preparation we have measured the differential expression of each PBP under β-lactam exposure via qRT-PCR and we haven't found significant differences. As opposed to PBP induced expression after exposure to sub-MIC concentrations observed in other bacterial species, for example S. aureus, P. aeruginosa PBP expression appears to be dependent on the growth phase and not significantly altered upon antibiotic exposure.
2) AmpC hyperexpression caused noticeable MIC increases for some betalactams but not others. Does this pattern agree with the different activities of AmpC on these beta-lactams in previous biochemical studies?
The results from our present study are in agreement with previous studies, according to the MIC changes observed in our work and the β-lactamase activity studied in previous works (now referenced in the main text). PBP occupancy reduction for the antipseudomonal penicillins and ceftazidime (PBP1a, 1b and 3), cefepime (PBP1a and 1b) and aztreonam (PBP3) correlated with MIC changes and previous observations as specific β-lactamase activity and kinetic hydrolysis profile measurements.
3) Although all these PBPs bind to beta-lactams, their contributions to P. aeruginosa survival vary. Do the results in this paper suggest any PBPs are more valuable antibiotic targets than others? Do they agree with previous studies on these PBPs, including both the in vitro IC50s using purified proteins (if available) and whether they are essential?
The results obtained in this paper, confirm what has already been observed in We refer to PBP binding affinities taking in account all the receptors, i.e., drugs that possess the same PBP binding profile and PBP selectivity (affinity for a defined group of PBPs). The sentence has been modified in the manuscript. Table 1, footnote a, the listing of 'PBP4' in the knockout strains is out of order compared with the columns in the table.

5)
The order has been changed accordingly.

Reviewer #3 (Comments for the Author):
The authors compare inhibition of binding of a fluorescent penicillin to P.
aeruginosa PBPs by a set of representative β-lactam antibiotics and βlactamase inhibitors. The experimental design unfortunately limits the conclusions that can be drawn and some revision is required to take these limitations into account. The experiments would have been better performed with different exposure times and inhibitor concentrations to assess kinetics. In this respect, the experiments with the BLIs, performed at the same concentration, are more interesting. It would be good to use a separate panel in Fig. 1, with a more useful scale, for this data set.
We agree with the reviewer, and we have modified the text accordingly. We decided to use 1/2 MIC concentration of all the studied drugs to relate PBP occupancy levels with the extracellular drug concentration, and afterwards to assess if the differences in MIC of the intrinsic or acquired resistance mechanisms could be related to changes in PBP occupancy. As this procedure is very time consuming, our priority was to determine the feasibility of the method and to assess if it would be sensitive to observe differences in target binding in the presence or absence of resistance determinants. Our next step will be to improve the method to for high throughput screening and to determine the binding kinetics.
We have changed the scale on the BLI panel in Fig. 1 as suggested by the reviewer.
(2) Line 165. Has the Bocillin FL assay been calibrated for P.
aeruginosa PBPs? Are the chosen concentration and incubation time appropriate to saturate all the PBPs? In the absence of this information it is difficult to interpret the data presented in Fig. 3. It appears that, for a particular PBP, the degree of labelling is similar in the different strains (except ΔdacB) but comparison of levels between PBPs is uncertain. For example, what is the significance of the difference between PBP1b and PBP2 labelling? Are the different levels really due to different amounts of protein or is the labelling of PBP2 less complete because of slower reaction or lower affinity?
While we certainly didn't reach steady-state conditions due to much higher enzyme concentrations than the substrate (Bocillin FL). Our assay has been previously calibrated for incubation time (5, 10, 15, 30, 90 mins), protein concentration (0.2, 0.5, 0.75, 1, 2, 4 mg/mL) and bocillin concentration (2.5, 5, 10, 15, 25 μM). In all the conditions tested, the differences observed for band intensity of PBP1b and 2, as an example, were always constant. We are aware of the substantially lower PBP2 acylation rate constant compared with PBP3 as Journal of Bacteriology 1996, however no such information is available for P. aeruginosa. Given the limitations of the assay we wanted to highlight that band signals were not altered in the different isogenic mutants; it was not our intention to compare band intensities of different PBPs for a given strain. We have changed the text in the manuscript to highlight our goal.
(3) Line 204. The assay used does not provide information about affinity: it is only possible to say that the reaction had a greater or a lesser extent in the particular reaction conditions used for each inhibitor and with each PBP. The rates of entry and reaction as well as affinity for target all play a role in the extent of inhibition but separating these requires a more comprehensive approach, as outlined above. Reference to affinity should be deleted throughout.
We agree with the reviewer comments, and we have changed the text accordingly.
(4) L. 378. There is no "mechanistic data", in terms of descriptions of rates and affinities, and this statement should be modified.
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